The broad aim of this research is to study the mechanisms by which hormones regulate enzyme levels in mammalian tissues. These studies involve the enzyme P-enolpyruvate carboxykinase, which plays a key role in both hepatic and renal gluconeogenesis. Measurements of enzyme turnover have indicated that the levels of P-enolpyruvate carboxykinase present in both liver and kidney, are regulated by changes in enzyme synthesis. During the next granting period I propose to study: (1) The hormonal regulation of the levels of mRNA coding for P-enolpyruvate carboxykinase in the cytosol of rat liver and kidney cortex. (2) Factors regulating the turnover of specific P-enolpyruvate carboxykinase mRNA. (3) Isolation and quantitation of mRNA for P-enolpyruvate carboxykinase in rat liver by two methods: a. Immunochemical isolation of polysomes using the dual-antibody precipitation technique. b. Isolation of mRNA using sucrose gradients, and the denaturing gel electrophoresis; all techniques involving conventional separation procedures. c. To clone the cDNA made from the isolated PEPCK mRNA to provide a molecular probe for DNA-RNA hybridization studies.